Sialic acids estimation

Ashraf, J., Butterfield, D. A., Jamefelt, J., and Laine, R. A. (1980). Enhancement of the Yu and Ledeen gas—liquid chromatographic, method for sialic acid estimation Use of methane chemical ionisation mass fragmentography. J. Lipid Res. 21, 1137-1141. 

A further method for estimation of sialic acid using periodic acid has been reported by Massamiri and coworkers.121 Fonnaldehyde produced by mild, oxidative cleavage of the D-en/f/iro-glycerol-l-yl side-chain of Neu can be detected by 3-methyl-2-benzothiazolinone hydrazone, which yields a green-blue color (absorbance maximum 625 nm). This test, which is slightly more sensitive than the thiobarbi-... 

A plot of the percentage amplification in the relative concentration (as estimated from relative peak area) of each aldol product over controls is shown in Figure 29.4. The peak assigned to sialic acid contributed approximately 40% of the total aldol product peak area after 160 hours in the presence of WGA but only 22% of the total aldol product peak area in the control mixture after the same time. This difference corresponds to a relative amplification of about 80%. Conversely, the contribution of KDN to the total aldol product peak area after 160 hours was 31% in the control incubation but only 6% in the presence of WGA this difference corresponds to a relative suppression of 80%. The observation that KDN and KDO were not suppressed to the same extent might be a consequence of WGA having a weak binding affinity for KDO. 

Two approaches have been proposed to improve the electrophoretic separation between bone and liver ALPs. Both methods exploit differences in the carbohydrate portions of the two forms of ALP. In one, electrophoresis is carried out in the presence of wheat germ lectin, which retards bone ALP migration more than the liver enzyme migration. In the other, serum is treated briefly (i.e.> for 15 min at 37 °C), with neuraminidase to remove part of the terminal sialic acid residues. As the sialic acid residues of bone ALP are more readily attacked than those of liver ALP, the electrophoretic mobility of the bone form is reduced more than that of liver ALP. The improved separation allows quantitative estimates to be made by densitometric scanning (Figure 21-5). ... 

Sampathkumar, S.-G., Jones, M. B., Yarema, K. J. (2006). Metabolic expression of thiol-derivatized sialic acids on the cell surface and their quantitative estimation by flow cytometry. Nat. Protocols, 1, 1840-1851. 

Finally, for the quantitative estimation of the 0-acyl content of sialic-acid-containing samples (Hestrin assay), also a colorimetric assay is routinely used. The method is based on the reaction with hydroxylamine in alkaline medium yielding hydroxamates, which form with Fe(C104)3 red chromophores, the absorbance of which is measured at 520 run [262]. 

Two types of reaction yield formyl pyruvic acid these are some P-eliminations (see below) and the periodate oxidation of most sialic acids. In each case the formyl pyruvate produced can be made to react with thio-barbituric acid to give an intensely coloured product, so giving the means of following P-eliminations and estimating very small amounts (1-2 (jig) of sialic acid. 

Brief oxidation with 0.025 M-sodium metaperiodate at pH 7, followed by estimation with the resorcinol or thiobarbituric acid reagent, has been used in the simultaneous assay of free and ketosidically bonded sialic acids, respectively." Treatment of 2-acetamido-2-deoxy-3,4 5,6-di-O-isopropylidene-aW liydo-D-glucose (192) or the analogous D-mannose derivative with alkali gave 2-acetamido-2,3-dideoxy-5,6-0-isopropylidene-a-D-eryt/iro-hex-2-enofuranose (193), which afforded the methyl 2-enofuranosides (194) with acidified methanol-acetone (194)... 

Such materials may interfere with the determination of hexosamines (see Chapter VIII). With the colors produced by sialic acid as a reference, Werner and Odin 100) have estimated the amounts of sialic acid in several glycoproteins, including serum glycoprotein and ovomucin. They used Bial s orcinol, the direct Ehrlich reaction, diphenylamine, and tryptophan-perchloric acid. 

The DMB-labeled sialic acids are applied to the HPLC equipped with a TSK-gel ODS-120T column (250 x 4.6 mm i.d., Tosoh), and a fluorescence detector (FP-2025, JASCO). The column is equilibrated using acetonitrile/methanol/water (9 7 84 by volume) at 26°C. Then 2-20 pL of the supernatants are applied to HPLC analysis for an isocratically flow at 1.0 mL/min and the DMB-labeled sialic acid is detected with a fluorescence detector at an excitation of 373 nm and an emission at 448 mn (Fig. 4). The estimate of the ratios of the quantity of internal sialic acid residues (C9(Sia)) to that of total sialic acid residues (C7(Sia) + C9(Sia)) can then be obtained. 

Svennerhohn L (1957) Quantitative estimation of sialic acids. II. A colorimetric resorcinol-hydrochloric acid method. Biochim Biophys Acta 24 604-611... 

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